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991.
S Schneider M G Wubbolts G Oesterhelt D Sanglard B Witholt 《Biotechnology and bioengineering》1999,64(3):333-341
Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450BM-3 monooxygenase [E.C. 1. 14.14.1], multiple oxidations of saturated and unsaturated fatty acids were performed by the enzyme under conditions of excess oxygen. The amount of oxygen dissolved in the culture medium strongly influenced the regioselectivity of the reaction, as reflected in the distribution and amount of oxidised products. We have verified by gas chromatography/mass spectrometry that the products of in vivo biotransformation of pentadecanoic acid by cytochrome P450BM-3 are identical to those formed in cell-free extracts containing the enzyme. The formation of keto- and dihydroxy acids, side products which are characteristic for in vitro conversions with purified cytochrome P450BM-3 in the presence of excess oxygen, has been observed as well. Thus, by varying the oxygen concentration, we could control the regioselectivity of oxidation and the number of products made. Under oxygen limiting conditions, only monooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained. Consequently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion. Furthermore, whole cell oxidation of two unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexadec-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids. Although we obtained higher activities of C15:0 conversion in vitro, the whole cell biocatalyst proved to be useful for specific oxidations of long-chain fatty acids since there is no need to add the costly cofactor NADPH. This biooxidation by E. coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonstrated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range. 相似文献
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H M Schneider M Loos 《Virchows Archiv. B, Cell pathology including molecular pathology》1978,29(3):225-228
The localization of amyloid P-components is demonstrated by immunofluorescence microscopy in normal human tissue (kidney, spleen, liver). The relation to collagen and to amyloidosis is discussed. 相似文献
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H. Schneider 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1965,35(1-2):37-40
Ohne ZusammenfassungHerrn Professor Dr. Dr. h. c.G. Becker zum 60. Geburtstag. 相似文献
1000.
Domain Structure of the ATP-Binding-Cassette Protein MalK of Salmonella typhimurium as Assessed by Coexpressed Half Molecules and LacK′-′MalK Chimeras 下载免费PDF全文
ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of Salmonella typhimurium and Escherichia coli is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of S. typhimurium. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a malK strain and displayed substantial regulatory activity. The same regulatory activity was obtained when malKC1 was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK. 相似文献